Distribution of Oxycodone in Postmortem Fluids and Tissues

DOT/FAA/AM-10/11 Office of Aerospace Medicine Washington, DC 20591

Sabra R. Botch Robert D. Johnson Arvind K. Chaturvedi Russell J. Lewis

Civil Aerospace Medical Institute Federal Aviation Administration Oklahoma City, OK 73125

June 2010

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1. Report No. 2. Government Accession No. 3. Recipient's Catalog No. DOT/FAA/AM-10/11

4. Title and Subtitle 5. Report Date Distribution of Oxycodone in Postmortem Fluids and Tissues June 2010 6. Performing Organization Code

7. Author(s) 8. Performing Organization Report No. Botch SR, Johnson RD, Chaturvedi AK, Lewis RL 9. Performing Organization Name and Address 10. Work Unit No. (TRAIS) FAA Civil Aerospace Medical Institute P.O. Box 25082 11. Contract or Grant No. Oklahoma City, OK 73125 12. Sponsoring Agency Name and Address 13. Type of Report and Period Covered Office of Aerospace Medicine Federal Aviation Administration 800 Independence Ave., S.W. Washington, DC 20591 14. Sponsoring Agency Code 15. Supplemental Notes This work was accomplished under the approved task AM-B-10-TOX-204. 16. Abstract Introduction: Oxycodone is a heavily used and abused analgesic agent. Its pharmacological effects, including euphoria, respiratory depression, nausea, and drowsiness, have the potential to adversely affect performance. The postmortem distribution of oxycodone has not been well characterized, particularly at sub-lethal levels. Therefore, an attempt was made to evaluate the distribution of oxycodone in postmortem specimens collected from aviation accidents.

Methods: A search of our database identified 4 oxycodone-positive fatalities from separate civil aviation accidents that occurred during a period of 6 years that had numerous biological tissues and fluids available (blood, urine, vitreous humor, liver, kidney, skeletal muscle, lung, spleen, heart muscle, and brain). These specimens were extracted using solid-phase extraction and were analyzed for oxycodone by GC/MS.

Results: Oxycodone concentration ranges (µg/mL, µg/g) found in the different tissues and fluids were: blood 0.027-0.742, urine 2.20 - 12.5, vitreous humor 0.048 - 0.118, liver 0.103-3.35, lung 0.047-1.35, kidney 0.045- 3.12, spleen 0.115-2.43, muscle 0.017-0.400, brain 0.032-1.36, and heart 0.038-3.19.

Conclusion: The blood concentrations found indicate that the oxycodone in these cases ranged from therapeutic to above therapeutic, but all were below lethal levels. Tissue/fluid to blood distribution coefficients were found to have large coefficients of variation (ranging from 26-128%), thereby rendering them unreliable for estimating a blood oxycodone concentration from a tissue value when no blood is available for analysis.

17. Key Words 18. Distribution Statement Forensic Toxicology, Oxycodone, Document is available to the public through the Distribution, GC/MS, Defense Technical Information Center, Ft. Belvoir, Aircraft Accident Investigation VA 22060; and the National Technical Information Service, Springfield, VA 22161

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price Unclassified Unclassified 9 Form DOT F 1700.7 (8-72) Reproduction of completed page authorized

Contents

Introduction...... 1 Materials and Methods ...... 1 . chemicals and Reagents...... 1 Gas Chromatographic/Mass Spectroscopic Conditions...... 1 . sample Selection and Storage...... 2 . calibrator and Control Preparation...... 2 . sample Preparation and Extraction Procedure ...... 2 Results and Discussion...... 3 References...... 4

iii

Distribution of Oxycodone in Postmortem Fluids and Tissues

Introduction Materials and Methods

Oxycodone, a semi-synthetic opioid agonist, is avail- Chemicals and Reagents able in several formulations, including controlled release All aqueous solutions were prepared using double (OxyContin®), immediate release (OxyIR®, OxyFast®), deionized water (DDW), which was obtained using a or in combination with other non-narcotic analgesics water purification system (Elga Water. systems;. high such as acetaminophen (Percocet®, Tylox®, Roxicet®) Wycombe, UK) ..all chemicals used were purchased in and aspirin (Percodan®) ..It is used for the treatment of the highest possible purity and used without any fur- postoperative pain, pain associated with cancer, and ther purification .. oxycodone and oxycodone-d3 were other pain management 1-3. Oxycodone is commonly obtained from. cerilliant. corporation (Round. rock, employed as a valuable alternative to morphine due to TX) as methanolic standards at concentrations of 1 .00 its longer analgesic action 4,5. Abuse of this semi-synthetic mg/mL and 0 .100 mg/mL, respectively, in sealed glass opiod and its role in fatal overdoses has been documented ampoules .. Propionic anhydride was obtained from in the literature 6-11. In addition to the analgesic effects, Sigma-Aldrich (St ..louis, MO) ..the pH of all solutions numerous side effects have been associated with the use was measured using a Corning Life Sciences model 430 of oxycodone ..dizziness, drowsiness, headache, nausea, pH meter (Acton, MA), connected to a Corning 3-in-1 vomiting, and muscle weakness have been linked with model pH electrode . the use of this drug .. Oxycodone is readily absorbed after oral administra- Gas Chromatographic/Mass Spectroscopic Conditions tion and has pharmacokinetics that differs from that of Analyses were performed using a bench-top gas chro- morphine 5. The volume of distribution (Vd) of oxyco- matograph/mass spectrometer (GC/MS), which consisted done is 277 ± 187 L/kg and it is a highly protein-bound of a Hewlett Packard (HP) 6890 series GC, interfaced with drug (45%) 4,12,13. The large Vd for oxycodone suggests a HP 5973 quadrupole MS (Agilent; Palo Alto, CA) ..the substantial distribution among all tissues and fluids in GC/MS was operated with a transfer line temperature of the body ..the half-life (t1/2) for the controlled-release 280°C and a source temperature of 250°c ..the MS was oral formulation of oxycodone has been documented tuned on a daily basis using perfluorotributylamine ..the to range from approximately 3 .5 to 8 hours,1,4,5 and electron multiplier voltage was set at 106 eV above the peak therapeutic plasma concentrations typically oc- tune value ..chromatographic separation was achieved cur within 1 to 3 hours, depending on inter-individual . using a Varian. factorFour crosslinked 100% methyl variation 14-16. The duration of action of immediate release siloxane capillary column 12 m x 0 .2 mm i .d ., 0 .33 µm oxycodone is ~4 hours .5 Up to 19% of ingested oxyco- film thickness (Varian Co .; Harbor City,.ca .) ..helium done is excreted unchanged after oral administration 1. was employed as the carrier gas at a flow rate of 1 .0 mL/ Within 24 hours, 30 to 60% of the original dose has min ..an HP 6890 autosampler was used to inject 1 µL been reported to be excreted in the urine as free and/ of extract into the GC/Ms ..the GC was equipped with or conjugated oxycodone, conjugated oxymorphone, a split/splitless injection port operated at 250°C in the and noroxycodone .14 splitless mode with the purge time of 0 .5 min ..the oven Scientific information concerning the distribution of temperature profile employed for the oxycodone analysis oxycodone in various body compartments is limited .7,9 was: 130°C – 240°C @ 20°C/min, 240°C – 244°C @ Postmortem samples from aviation accident victims are 2°C/min, 244°C – 290°C @ 40°C/min, with a final hold analyzed for drugs at the Federal Aviation Administra- time of 1 35. min ..Quantitation and qualifier ions for each tion’s. civil. aerospace Medical Institute (CAMI) .. a. analyte were then selected based on both abundance and search of our laboratory accident database identified four mass-to-charge ratio (m/z) ..to increase reproducibility and aviation fatalities that were positive for oxycodone in reduce interference, high-mass ions were selected when blood ..these cases also had a full complement of other possible ..the ions chosen for oxycodone were 371 (quant), tissue and fluid types available for additional analysis .. 314, 372, and for oxycodone-D3 were 374 (quant), 317, Therefore, this study was pursued to determine the 375 ..upon selection of unique ions, the MS was run in distribution of oxycodone in postmortem tissues and selected ion monitoring (SIM) mode with a dwell time fluids of the four aviation accident victims .. of 30 msec for each recorded ion . 1 Sample Selection and Storage Sample Preparation and Extraction Procedure A search of the CAMI ToxFlo™ database (DiscoverSoft Postmortem specimens, calibrators, and controls were Development,. llc;. oklahoma. city,. oK) identified extracted in the following manner ..tissue specimens were four oxycodone-positive fatalities from separate civil homogenized in DDW (1 .00% NaF) with a 1:2 dilution aviation accidents that occurred during a period of 6 tissue:DDW, using an Omni post-mounted homogenizer years (2003-2008) ..these four cases had a majority of the (Marietta, GA) ..the generator used with this homogenizer desired biological tissues and fluids (blood, urine, vitreous was 30 mm in diameter and set to rotate at 22,000 rpm .. humor, liver, kidney, skeletal muscle, lung, spleen, heart Three-mL aliquots of fluid, calibrator, and control, and muscle, and brain) were used for the study ..In all cases, 3 .0 g aliquots of each tissue homogenate (1 .0 g tissue) blood was stored at -20°C in tubes containing 1 .00% were transferred to individual 16 x 150 mm screw-top (w/v) sodium fluoride/potassium oxalate until analysis .. tubes ..to each specimen, calibrator, and control, 1 .00 All other specimens were stored without preservation at mL of the internal standard 400 ng solution was added .. -20°C until analysis .. Samples were vortexed briefly and allowed to stand at room temperature for 10 min ..six mL of 0 .10 M phosphate Calibrator and Control Preparation buffer, pH 6 .00 was added to each sample, and the tubes Calibration curves for oxycodone were prepared by were then shaken by hand for 2 min ..centrifugation at serial dilution utilizing bovine whole blood as the dilu- 1230χg for 45 min provided removal of cellular debris ent ..calibrators were prepared from one set of original and proteins .. stock standard solutions, while controls were prepared The extracts were transferred to Bond Elute Certify® in a similar manner as calibrators but from a second set solid-phase extraction (SPE) columns (Varian Co ;. Harbor of unique stock solutions ..the calibration curve was pre- City,.ca ),. which had been pre-conditioned with 2 00. mL pared at concentrations ranging from 3 .13 – 800 ng/ml .. methanol, followed by 2 .00 mL water ..care was taken not A minimum of seven calibrators were used to construct to dry the column prior to adding the extract ..column the calibration curve ..controls were prepared at concen- flow rates of 1-2 mL/min were maintained in each SPE trations of 80 and 320 ng/mL and extracted with the step using a Varian 24 port Cerex SPE processor (Var- unknowns to verify the accuracy of the calibration curve ian Co .; Harbor City,.ca .) with a nitrogen pressure of 3 and the validity of the extraction ..the internal standard psi ..once each sample had passed through its respective solution, oxycodone-d3, was prepared at a concentration column, the columns were washed with 1 .00 mL of wa- of 400 ng/mL in DDW . ter, followed by 1 .00 mL of 0 .10 M sodium acetate, pH Quantitation was achieved via an internal standard 4 .00 ..the columns were again washed by adding 2 .00 calibration procedure ..response ratios for each compound mL methanol to each ..ollowing f the methanol wash, were determined for every sample analyzed ..the response the columns were dried completely with 25 psi nitrogen ratio was calculated by dividing the area of the analyte for 5 min ..the analytes were eluted off the columns with peak by the area of the internal standard peak ..calibra- 3 .00 mL of 2 .00% ammonium hydroxide in an 80:20 tion curves were derived by plotting a linear regression mixture of methylene chloride/isopropanol, which was of the analyte/internal standard response ratio versus prepared fresh daily ..eluents were evaporated to dryness the analyte concentration for each respective calibrator .. in a. turboVap Concentration Workstation at 40°C These calibration curves were then used to determine (Caliper Life Sciences; Hopkinton, MA) under a stream of the concentrations of each compound in the prepared dry nitrogen ..the procedure used is a standard analytical controls and biological specimens ..acceptability criteria procedure of our laboratory for multi-opiate analysis, re- employed for analyte identification and quantitation were quiring derivatization ..therefore, even though oxycodone as follows: 1) ion ratios for a given analyte, measured as may be analyzed without derivatization, the derivatization the peak area of a qualifier ion divided by the peak area step was performed so that analytical parameters could of the quantitation ion, were required to be within ± be directly compared to those that had been previously 20% of the average of the ion ratios for each respective obtained ..the derivatization was accomplished by adding calibrator used to construct the calibration curve for that 50 µL of pyridine, followed by 50 µL of propionic anhy- analyte; 2) each ion monitored was required to have a dride to each specimen tube ..the samples were capped minimum signal-to-noise ratio (S/N) of 10; and 3) the tightly, vortexed, and incubated at 40°C for 60 min ..the analyte was required to have a retention time within ± tubes were allowed to cool to room temperature, and 2 .00% of the average retention time for each respective the contents were evaporated to dryness under a stream calibrator used to construct the calibration curve for that of dry nitrogen in a TurboVap set at 40°c ..once dry, analyte ..analytes not meeting these criteria were reported the contents of each tube were reconstituted in 50 µL of as either negative or inconclusive .. ethyl acetate and transferred to GC/MS vials for analysis . 2 Results and Discussion The injection of an ethyl acetate blank following the 800 ng/mL calibrator showed no carryover contamination .. The procedure used for the analysis of oxycodone was Subsequently, ethyl acetate blanks were utilized between reproducible and sensitive ..oxycodone and oxycodone- each postmortem specimen throughout the sample se- d3 chromatographic peaks suffered no interference from quence to verify that no sample-to-sample contamination endogenous/exogenous matrix components ..the mass had occurred . spectra of oxycodone and oxycodone‑d3 provided numer- Whole blood oxycodone concentrations found in the ous high mass ions ..It should be noted that an isotope ion four cases examined ranged from 0 .027 to 0 .742 mg/mL was used as one of the qualifier ions for both oxycodone and were within 10% of the value originally determined .. and oxycodone-d3 ..this approach was necessary due to This finding verified that no deterioration in oxycodone cross-contribution between the analyte and oxycodone- blood concentration had occurred during storage ..thera- d3 when other ions were evaluated ..the linear dynamic peutic concentrations of oxycodone in whole blood range range (LDR), limit of detection (LOD), and limit of from 0 .010 to 0 .100 mg/ml 17. Toxic levels of oxycodone quantitation (LOQ) for oxycodone were determined have been reported to be 0 .200 - 5 .000 mg/mL and vary while utilizing bovine whole blood as the matrix ..the greatly .17 Blood concentrations in these cases could generally LDR were determined to be 3 .13 – 800 ng/ml ..cor- be above therapeutic and neared possibly toxic levels ..the relation coefficients for the calibration curves used were concentration of oxycodone in each postmortem specimen greater than 0 .997 when a weighting factor of 1/X was from these four cases is given in Table 1 ..With a relatively employed ..the LOD was defined as the lowest analyte large Vd (277 ± 186 L/kg),4 oxycodone was expected to be concentration detectable that met the above-discussed found at high concentrations in the tissues analyzed, which identification criteria ..the LOQ was defined as the low- was consistent with our findings ..the mean concentrations est analyte concentration detectable that not only met (µg/mL, µg/g) of oxycodone found were: blood 0 .408 all identification criteria, as discussed above, but also (0 .027-0 .742, n=4); urine 7 .36 (2 .20 and 12 .5, n=2); had an experimentally determined concentration within vitreous humor 0 .083 (0 .048 and 0 .118, n=2); liver 1 .46 ± 20% of its prepared value ..the LOD for oxycodone (0 .103-3 .35, n=4); lung 0 .477 (0 .047-1 .35, n=4), kidney was determined to be 1 .56 ng/ml ..the LOQ for this 0 .991 (0 .045-3 .12, n=4), spleen 1 .10 (0 .115-2 .43, n=4); compound was determined to be 3 .13 ng/ml . muscle 0 165. (0 017-0. 400,. n=4); brain 0 437. (0 032-1. 36,. Carryover was not found to be a problem on the GC/ n=4); and heart 0 .900 (0 .038-3 .19, n=4) ..the distribution MS; however, it was initially investigated and subsequently coefficients for oxycodone, expressed as specimen type/blood monitored by the use of ethyl acetate blank injections .. ratio ± SD, are summarized in Table 2 ..the oxycodone

Table 1. Oxycodone concentrations obtained from 4 pilot fatalities.*

Case Blood Urine VH† Liver Lung Kidney Spleen Muscle Brain Heart 1 0.317 12.5 —‡ 3.35 1.35 0.312 2.43 0.400 1.36 3.19 2 0.741 — — 2.12 0.315 0.638 1.58 0.157 0.220 0.273 3 0.027 — 0.048 0.103 0.047 0.045 0.115 0.017 0.033 0.038 4 0.137 2.20 0.119 0.403 0.195 0.155 0.272 0.085 0.136 0.099 * All concentrations shown in units of g/mL or g/g † Vitreous Humor ‡ Specimen type not available for analysis

Table 2. Postmortem tissue distribution coefficients for oxycodone. Urine/ VH*/ Liver/ Lung/ Kidney/ Spleen/ Muscle/ Brain/ Heart/

Blood Blood Blood Blood Blood Blood Blood Blood Blood n 2 2 4 4 4 4 4 4 4 Mean 27.8 1.32 5.04 1.96 1.16 4.00 0.68 1.69 3.14 s.d. † 11.7 0.45 3.22 1.41 0.30 2.30 0.38 1.53 4.02 CV‡ 42 34 64 72 26 57 55 90 128 * Vitreous Humor † Standard deviation ‡ Coefficient of variation 3 distribution coefficients for these specimen types had coef- References ficient of variation (CV) values between 26 and 128% .. Although blood drug concentrations may aid in de- 1 .. Brunton,.l .l ., Lazo, J .s, . and Parker, K .l ., Eds .. termining impairment and/or cause or manner of death, Goodman & Gilman’s The Pharmacological Basis the availability of blood samples from aviation accident of Therapeutics, Eleventh ed ..(McGraw-Hill, New fatalities may not be always an option ..this limitation York, 2006) . is primarily due to the destructive nature of aviation accidents on the human body ..In fact, the FAA laboratory 2 ..uutinen, n .l .s, . Wuolijoki,.e ., and Pentikainen, receives blood from only approximately 70% of the fatal I .t ..diclofenac and Oxycodone in Treatment of cases, and the majority of the blood samples are collected Postoperative Pain:. a. double-Blind. trial .. Acta from the chest cavity ..such blood samples can not only Anaesthesiol Scand, 30: 620-4 (1986) . readily experience postmortem redistribution, but they 3 ..e d Conno,.f ., Ripamonti,.c ,. Sbanotto,.a ., et al ..a. can easily be contaminated with drug(s) from ruptured Clinical Study on the Use of Codeine, Oxycodone, organs ..therefore, caution must be exercised when using Dextropropoxyphene, Buprenorphine, and Pen- drug values obtained from chest cavity blood . tazocine in Cancer Pain ..J Pain Symptom Manage, Based upon the present study, an attempt was made to 6: 423-7 (1991) . establish distribution coefficients for oxycodone between the various tissue/fluids and blood ..those coefficients 4 .. Poyhia,.r ., Seppala,.t ., Olkkola, K .t ., et al ..the might be useful to potentially estimate a blood concen- Pharmacokinetics and Metabolism of Oxycodone tration in cases where blood is not available ..though After Intramuscular and Oral Administration to the CV values varied considerably (Table 2), the results Healthy Subjects ..Br J Clin Pharmacol, 33: 617-21 obtained from the limited number of cases suggest that (1992) . oxycodone blood concentrations cannot be estimated 5 ..ugo, l .r .a ..and Kern,.s .e ..the Pharmacokinetics from any of the tissue types examined .the large range of Oxycodone ..J Pain Palliat Care Pharmacother, in distribution values within a given specimen/blood 18: 17-30 (2004) . ratio could be due to the postmortem redistribution of the drug with respect to the different postmortem inter- 6 .. Wolf, B .c ., Lavezzi, W .a ., Sullivan,.l .M ., et al .. vals, the blood collection sites, and/or presence of other One Hundred Seventy-Two Deaths Involving the drugs 18. It might also be affected by whether the victim Use of Oxycodone in Palm Beach County ..J Forensic was a chronic or an acute user of oxycodone ..It is well Sci, 50: 192-5 (2005) . established that the postmortem redistribution of drugs 7 .. anderson,.d .t ., Fritz, K .l, . and Muto, J .J ..oxy- is more pronounced with basic drugs as they have large contin: The. concept of a “Ghost Pill” and the apparent volumes of distribution ..for oxycodone, the Postmortem Tissue Distribution of Oxycodone in Vd is 277 ± 187 l/kg 4,12,13. In spite of these limitations 36 Cases ..J Anal Toxicol, 26: 448-59 (2002) . and variations, it is still important to gain knowledge of 8 ..rummer, d .o .h, . Syrjanen, M .l, . Phelan, M ,. et al .. drug concentrations in tissues as these analytical values A Study of Deaths Involving Oxycodone ..J Forensic may help in differentiating therapeutic use of the drugs Sci, 39: 1069-75 (1994) . from their overdose 19. . additionally, such information will contribute to the limited tissue distribution data 9 ..piller, s .h .a Postmortem.. Oxycodone and Hydro- available in the literature for this drug ..however, one has codone Blood Concentrations ..J Forensic Sci, 48: to be careful when drawing final conclusions from this 429-31 (2003) . data because the blood collection sites and postmortem 10 ..hompson, t J G. ,. Vanderwerf,.s ,. Seningen, J ,. et al .. intervals were often not known or not established .. Free Oxycodone Concentrations in 67 Postmortem Cases from the Hennepin County Medical Exam- iner’s Office ..J Anal Toxicol, 32: 673-9 (2008) . 11 .. cone,.e .J ., Fant,.r .V ., Rohay, J .M ., et al ..oxycodone Involvement in Drug Abuse Deaths II ..evi- dence for Toxic Multiple Drug-Drug Interactions .. J Anal Toxicol, 28: 616-24 (2004) .

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